The drug aphidicolin inhibits the reproduction of vaccinia virus. DNA synthesis in uninfected cells is reduced by the interaction of aphidicolin with DNA polymerase Alpha. The DNA polymerase coded by vaccinia virus is inhibited by the drug, but it is less sensitive than DNA polymerase Alpha. At drug concentrations greater than 40 MuM both plaque formation and viral yield show a rapid exponential decrease. At 20 MuM there is only a slight decrease in the number of plaques, but they are reduced in size. Introduction of the drug 2 hr. prior to infection, at infection, or 1.5 hr. after infection does not significantly alter the inhibition of viral yield. Also, the effect of the drug on viral yield is completely reversible for at least 12 hr. after infection. Aphidicolin reduces virus formation by reducing the rate of viral DNA synthesis. With 20 MuM drug present for 6 hr. after infection, viral DNA synthesis is reduced to 12% of control values. Viral DNA synthesis is returned to the normal rate by simply washing and suspending infected cells with drug-free medium. A mutant vaccinia virus which is resistant to aphidicolin has been developed. The mutant will produce substantial amounts of progeny in the presence of 120 MuM drug, the highest concentration tested. In vitro tests of viral DNA polymerase isolated from cells infected with wild-type and resistant virus indicate that the enzyme from the mutant is more resistant to the drug than the polymerase from wild-type virus. The aphidicolin resistant mutant is not resistant to phosphonoacetate which also interacts with the viral DNA polymerase.